Over the past 20 years, the fields of synthetic biology and synthetic biosystems engineering have grown into mature disciplines, leading to significant breakthroughs in cancer research, diagnostics, cell-based medicines, biochemical production, etc. Application of mathematical modelling to biological and biochemical systems have not only given great insight into how these systems function, but also have lent enough predictive power to aid in the forward-engineering of synthetic constructs. However, progress has been impeded by several modes of context-dependence unique to biological and biochemical systems that are not seen in traditional engineering disciplines, resulting in the need for lengthy design-build-test cycles before functional prototypes are generated.In this work, two of these universal modes of context dependence – resource competition and growth feedback –their effects on synthetic gene circuits and potential control mechanisms, are studied and characterized. Results demonstrate that a novel competitive control architecture can be utilized to mitigate the effects of winner-take-all resource competition (a form of context dependence where distinct gene modules influence each other by competing over a shared pool of transcriptional/translational resources) in synthetic gene circuits and restore circuits to their intended function. Application of the fluctuation-dissipation theorem and rigorous stochastic simulations demonstrate that realistic resource constraints present in cells at the transcriptional and translational levels influence noise in gene circuits in a nonmonotonic fashion, either increasing or decreasing noise depending on the transcriptional/translational capacity. Growth feedback on the other hand links circuit function to cellular growth rate via increased protein dilution rate during exponential growth phase. This in turn can result in the collapse of bistable gene circuits as the accelerated dilution rate forces switches in a high stable state to fall to a low stable state. Mathematical modelling and experimental data demonstrate that application of repressive links can insulate sensitive parts of gene circuits against growth-fluctuations and can in turn increase the robustness of multistable circuits in growth contexts. The results presented in this work aid in the accumulation of understanding of biological and biochemical context dependence, and corresponding control strategies and design principles engineers can utilize to mitigate these effects.

Clustered regularly interspace short palindromic repeats (CRISPR) and CRISPR associated (Cas) technologies have become integral to genome editing. Canonical CRISPR-Cas9 systems function as a ribonucleic acid (RNA)-guided nucleases. Single guide RNAs (sgRNA) can be easily designed to target Cas9’s nuclease activity towards protospacer deoxyribonucleic acid (DNA) sequences. The relatively ease and efficiency of CRISPR-Cas9 systems has enabled numerous technologies and DNA manipulations. Genome engineering in human cell lines is centered around the study of genetic contribution to disease phenotypes. However, canonical CRISPR-Cas9 systems are largely reliant on double stranded DNA breaks (DSBs). DSBs can induce unintended genomic changes including deletions and complex rearrangements. Likewise, DSBs can induce apoptosis and cell cycle arrest confounding applications of Cas9-based systems for disease modeling. Base editors are a novel class of nicking Cas9 engineered with a cytidine or adenosine deaminase. Base editors can install single letter DNA edits without DSBs. However, detecting single letter DNA edits is cumbersome, requiring onerous DNA isolation and sequencing, hampering experimental throughput. This document describes the creation of a fluorescent reporter system to detect Cytosine-to-Thymine (C-to-T) base editing. The fluorescent reporter utilizes an engineered blue fluorescent protein (BFP) that is converted to green fluorescent protein (GFP) upon targeted C-to-T conversion. The BFP-to-GFP conversion enables the creation of a strategy to isolate edited cell populations, termed Transient Reporter for Editing Enrichment (TREE). TREE increases the ease of optimizing base editor designs and assists in editing cell types recalcitrant to DNA editing. More recently, Prime editing has been demonstrated to introduce user defined DNA edits without the need for DSBs and donor DNA. Prime editing requires specialized prime editing guide RNAs (pegRNAs). pegRNAs are however difficult to manually design. This document describes the creation of a software tool: Prime Induced Nucleotide Engineering Creator of New Edits (PINE-CONE). PINE-CONE rapidly designs pegRNAs based off basic edit information and will assist with synthetic biology and biomedical research.

Recombinases are powerful tools for genome engineering and synthetic biology, however recombinases are limited by a lack of user-programmability and often require complex directed-evolution experiments to retarget specificity. Conversely, CRISPR systems have extreme versatility yet can induce off-target mutations and karyotypic destabilization. To address these constraints we developed an RNA-guided recombinase protein by fusing a hyperactive mutant resolvase from transposon TN3 to catalytically inactive Cas9. We validated recombinase-Cas9 (rCas9) function in model eukaryote Saccharomyces cerevisiae using a chromosomally integrated fluorescent reporter. Moreover, we demonstrated cooperative targeting by CRISPR RNAs at spacings of 22 or 40bps is necessary for directing recombination. Using PCR and Sanger sequencing, we confirmed rCas9 targets DNA recombination. With further development we envision rCas9 becoming useful in the development of RNA-programmed genetic circuitry as well as high-specificity genome engineering.